Use of Ginkgo extract

ABSTRACT

The present invention is directed to the use of the extract of  Ginkgo biloba  leaves or isolated Ginkgolide B (GKB), a component of the extract of  Ginkgo biloba  leaves in a method for decreasing the expression of peripheral-type benzodiazepine receptor (PBR) in cells of a patient in need thereof. Further, the present invention is directed to the use of the extract of  Ginkgo biloba  leaves or isolated GKB in a method for decreasing the proliferation of cancer cells in a patient. More particularly, the present invention is directed to the use of the extract of  Ginkgo biloba  leaves or isolated GKB in a method of decreasing cancer cell proliferation in a patient wherein the cancer cell is human breast cancer cell. Even more particularly, the present invention is directed to the use of the extract of  Ginkgo biloba  leaves or isolated GKB in method of decreasing cancer cell proliferation in a patient wherein the cancer cell is of the aggressive and invasive phenotype and expresses high levels of PBR in comparison to non-aggressive cancer cell.

This application claims the benefit of 60/148,604 filed Aug. 12, 1999.

BACKGROUND OF THE INVENTION

The present invention is directed to the use of the extract of Ginkgobiloba leaves or isolated ginkgolide B (GKB), a component of the extractof Ginkgo biloba leaves in a method for decreasing the expression ofperipheral-type benzodiazepine receptor (PBR) in cells of a patient inneed thereof. Further, the present invention is directed to the use ofthe extract of Ginkgo biloba leaves or isolated GKB in a method fordecreasing the proliferation of cancer cells in a patient in needthereof. More particularly, the present invention is directed to the useof the extract of Ginkgo biloba leaves or isolated GKB in a method ofdecreasing cancer cell proliferation in a patient in need thereofwherein said cancer cell is human breast cancer cells. Even moreparticularly, the present invention is directed to the use of theextract of Ginkgo biloba leaves or isolated GKB in a method ofdecreasing cancer cell proliferation in a patient in need thereofwherein said cancer cell are of the aggressive and invasive phenotypeand expresses high levels of PBR in comparison to non-aggressive cancercells.

In another aspect, the present invention is directed to the use ofextract of Ginkgo biloba leaves to decrease the expression ofthirty-five (35) gene products as is further detailed, hereinbelow.

It is preferred that a particular formulation of Ginkgo biloba leavesextract known as EGB 761® (a product of IPSEN, Paris, France) be aconstituent in a composition or used in a method of the presentinvention.

Ginkgo biloba is one of the most ancient trees and extracts from itsleaves have been used in traditional medicine for several hundred years.There are numerous studies describing the beneficial effects of Ginkgobiloba extracts on patients with disturbances in vigilance, memory, andcognitive functions associated with aging and senility, and on thosewith all types of dementias, mood changes, and the ability to cope withdaily stressors. A standardized extract of Ginkgo biloba leaves, termedEGB 761®, has been used in most of these studies. This extract is alsoknown to have cardioprotective effects (DeFeudis F. V. Ginkgo bilobaextract (EGB 761®): from chemistry to clinic. Ullstein Medical,Wisbaden, Germany. 400 pp. 1998; Tosaki, A., Droy-Lefaix, M. T., Pali,T., and Das, D. K., Free Rad. Biol. Med., 14: 361–370, 1993). Theseeffects have been attributed, at least in part, to the free radicalscavenging properties of EGb761®, probably due to the presence offlavonoid or terpenoid constituents in the extract. Recent in vivo andin vitro studies demonstrated that the terpene constituents of EGB 761®,ginkgolides and bilobalide, have anti-oxidant properties (Pietri, S.,Maurelli, E., Drieu, K., and Culcasi, M., J. Mol. Cell. Cardiol., 29:733–742, 1997; Yao, Z., Boujrad, N., Drieu, K., and Papadopoulos, V.,Adv. Ginkgo Biloba Res. 7: 129–138, 1998). Other studies of EGB 761®have reported medicinal value of the product in the treatment of avariety of clinical disorders including cerebrovascular and peripheralvascular insufficiencies associated with aging and senility. See e.g.,Ginkgo biloba Extract (EGB 761®) Pharmacological Activities and ClinicalApplications, DeFeudis, F. V., Eds, Elsevier, 1991; and Ullstein Medical1998, Ginkgo biloba extract (EGB 761®), Eds. Wiesbaden, DeFeudis, F. V.The extract contains 24% ginkgo-flavone glycosides, 6% terpene lactones(ginkgolides and bilobalide), about 7% proanthocyanidins and severalother constituents. See Boralle, N., et al., In: Ginkgolides, Chemistry,Biology, Pharmacology and Clinical perspectives, Ed: Braquet, P., J. R.Prous Science Publishers, 1988.

Tumor progression is a multi-step process in which normal cellsgradually acquire more malignant phenotypes, including the ability toinvade tissues and form metastases, the primary cause of mortality inbreast cancer. During this process, the “aberrant” expression of anumber of gene products may be the cause or the result of tumorigenesis.Considering that the first step of tumor progression is cellproliferation, it can be proposed that tumorigenesis and malignancy arerelated to the proliferative potential of tumoral cells.

Studies in a number of tumors such as rat brain containing glioma tumors(Richfield, E. K. et al. (1988) Neurology 38:1255–1262), colonicadenocarcinoma and ovarian carcinoma (Katz, Y. et al. (1988) Eur. J.Pharmacol. 148:483–484 and Katz, Y. et al. (1990) Clinical Sci.78:155–158) have shown an abundance of peripheral-type benzodiazepinereceptors (PBR) compared to normal tissue. Moreover, a 12-fold increasein PBR density relative to normal parenchyma, was found in human brainglioma or astrocytoma (Cornu, P. et al. (1992) Acta Neurochir.199:146–152). The authors suggested that PBR densities may reflect theproliferative activity of the receptor in these tissues. Recently, theinvolvement of PBR in cell proliferation was further shown (Neary, J. T.et al. (1995) Brain Research 675:27–30; Miettinen, H. et al. (1995)Cancer Research 55:2691–2695), and its expression of human astrocytictumors was found to be associated with tumor malignancy andproliferative index (Miettinen, H. et al. supra; Alho, H. (1994) CellGrowth Different. 5:1005–1014). Further studies have shown that PBRreceptors are abundant in human glioblastomas (Broaddus, W. C., et al.,Brain Research, Vol. 518:199–208, 1990; and Pappata, S., et al., J.Nuclear Med., 32:1608–1610, 1991).

PBR is an 18-kDa protein discovered as a class of binding sites forbenzodiazepines distinct from the GABA neurotransmitter receptor(Papadopoulos, V. (1993) Endocr. Rev. 14:222–240). PBR are extremelyabundant in steroidogenic cells and found primarily on outermitochondrial membranes (Anholt, R. et al. (1986) J. Biol. Chem.261:576–583). PBR is thought to be part of the multimeric complexcomposed of the 18-kDa isoquinoline-binding protein and the 34-kDapore-forming voltage-dependent anion channel protein, preferentiallylocated on the outer/inner mitochondrial membrane contact sites(McEnery, M. W. et al. Proc. Natl. Acad. Sci. U.S.A. 89:3170–3174;Garnier, M. et al. (1994) Mol. Pharmacol. 45:201–211; Papadopoulos, V.et al. (1994) Mol. Cel. Endocr. 104:R5–R9). Drug ligands of PBR, uponbinding to the receptor, stimulate steroid synthesis in steroidogeniccells in vitro (Papadopoulos, V. et al. (1990) J. Biol. Chem.265:3772–3779; Ritta, M. N. et al. (1989) Neuroendocrinology 49:262–266;Barnea, E. R. et al. (1989) Mol. Cell Endocr. 64:155–159; Amsterdam, A.and Suh, B. S. (1991) Endocrinology 128:503–510; Yanagibashi, K. et al.(1989) J. Biochem. (Tokyo) 106:1026–1029). Likewise, in vivo studiesshowed that high affinity PBR ligands increase steroid plasma levels inhypophysectomized rats (Papadopoulos V. et al (1997) Steroids 62:21–28).Further in vitro studies on isolated mitochondria provided evidence thatPBR ligands, drug ligands, or the endogenous PBR ligand, the polypeptidediazepam binding inhibitor (BDI) (Papadopoulos, V. et al. (1997)Steroids 62:21–28), stimulate pregnenolone formation by increasing therate of cholesterol transfer from the outer to the inner mitochondrialmembrane (Krueger, K. E. and Papadopoulos, V. (1990) J. Biol. Chem265:15015–15022; Yanagibashi, K. et al. (1988) Endocrinology 123:2075–2082; Besman, M. J. et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:4897–4901; Papadopoulos, V. et al. (1991) Endocrinology129:1481–1488).

Based on the amino acid sequence of the 18-kDa PBR, a three dimensionalmodel was developed (Papadopoulos, V. (1996) In: The Leydig Cell. Payne,A. H. et al. (eds) Cache River Press, IL, pp. 596–628). This model wasshown to accomodate a cholesterol molecule and function as a channel,supporting the role of PBR in cholesterol transport. Recently wedemonstrated the role of PBR in steroidogenesis by generating PBRnegative cells by homologous recombination (Papadopoulos, V. et al.(1997) J. Biol. Chem. 272:32129–32135) that failed to produce steroids.However, addition of the hydrosoluble analogue of cholesterol,22R-hydroxycholesterol, recovered steroid production by these cells,indicating that the cholesterol transport mechanism was impaired.Further cholesterol transport experiments in bacteria expressing the18-kDa PBR protein provided definitive evidence for a function as acholesterol channel/transporter (Li and Papadopoulos, V. et al., (1998)Endocrinology).

We hypothesized that the peripheral-type benzodiazepine receptor is partof the changes in cellular and molecular functions that account for theincreased aggressive behavior in cancer, and we chose to examine thishypothesis in human breast cancer. Breast cancer is the most commonneoplasm and the leading cause of cancer-related deaths for women inmost developing countries (Lippman, M. E. (1993) Science 259:631–632),affecting nearly 184,000 women, with over 46,000 deaths annually in theU.S. alone (American Cancer Society, 1996). Human breast cells areunlike brain and gonadal cells and cannot produce steroids, but likemany other cells in the body, are able to metabolize steroids.

Increased PBR expression correlates with increased aggressive behaviorof tumor cells. Invasive tumors invade and grow locally but they do notmetastasize. However, the aggressive tumors have the ability to invadeand metastasize through the blood vessels to different places of thehuman body. Tumor metastasis into vital organs (such as lungs) is themost common cause of death.

The correlation between high levels of expression of PBR and metastaticpotential in for human breast cancer is shown in copending U.S.application Ser. No. 09/047,652 filed Mar. 25, 1998, in which VassiliosPapadopoulos of the instant application is a co-inventor. However, dueto the involvement of PBR in cell proliferation, and the expression ofPBR in all cells, it is likely that this correlation would exist forother solid tumors and cancers such as prostate cancer, colon cancer,brain tumors, and tumors in steroid producing tissues such as gonadaltumors, to name a few.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method ofcombating cancer in a patient in need of such combating, wherein thecancer is caused by the deregulation of expression of proteins having arole in regulating tumor cells, which comprises administering aneffective amount of Ginkgo biloba extracts or isolated Ginkgolide B tosaid patient.

In another aspect, the present invention is directed to a method ofcombating the proliferation of cancer cells in a patient in need of suchcombating, wherein the proliferation is caused by the deregulation ofexpression of proteins having a role in regulating tumor cells, whichcomprises administering an effective amount of Ginkgo biloba extracts orisolated Ginkgolide B to said patient.

In another aspect, the present invention is directed to a method ofcombating the proliferation of cancer cells in a patient in need of suchcombating, wherein the proliferation is caused by over-expression ofproteins having a role in regulating tumor cells, which comprisesadministering an effective amount of Ginkgo biloba extracts or isolatedGinkgolide B to said patient.

In another aspect, the present invention is directed to a method ofcombating the proliferation of cancer cells having an aggressivephenotype in a patient in need of such combating, wherein theproliferation is caused by the over-expression of peripheral-typebenzodiazepine receptor protein, which comprises administering aneffective amount of Ginkgo biloba extracts or isolated Ginkgolide B tosaid patient.

In another aspect, the present invention is directed to a method ofcombating the proliferation of cancer cells, where the proliferation iscaused by the over-expression of oncogenes, by decreasing the expressionof said oncogenes in a patient in need of such combating, whichcomprises administering an effective amount of Ginkgo biloba extracts orisolated Ginkgolide B to said patient. A preferred method of theimmediately foregoing method is where said oncogenes are one or more ofAPC, PE-1, RhoA and c-Jun.

In another aspect, the present invention is directed to a method ofdecreasing the expression of peripheral-type benzodiazepine receptor incancer cells in a patient in need of such decreasing, wherein saidcancer cells express an abnormal level of peripheral-type benzodiazepinereceptor relative to normal cancer cells, which comprises administeringan effective amount of Ginkgo biloba extracts or isolated Ginkgolide Bto said patient. A preferred method of the immediately foregoing methodis where the cancer cells are human breast cancer cells; humanglioblastomas; human brain tumors; human astrocytomas; human coloniccarcinoma; human colonic adenocarcinoma; human ovarian carcinomas; andhuman hepatocellular carcinoma.

In another aspect, the present invention is directed to a method ofdecreasing the expression of peripheral-type benzodiazepine receptormRNA in cancer cells in a patient in need of such decreasing, whichcomprises administering an effective amount of Ginkgo biloba extracts orisolated Ginkgolide B to said patient.

In another aspect, the present invention is directed to a method ofincreasing the expression of c-Myc protooncogene in a patient in need ofsuch increasing, which comprises administering an effective amount ofGinkgo biloba extracts or isolated Ginkgolide B to said patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of cell cycle regulators prothymosin-α, CDK2,p55CDC, myeloblastin and p120 proliferating-cell nuclear antigen (PCNA)in a patient in need of such decreasing, which comprises administeringan effective amount of Ginkgo biloba extracts or isolated Ginkgolide Bto said patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of intracellular signal transductionmodulators NET1 and ERK2, in a patient in need of such decreasing, whichcomprises administering an effective amount of Ginkgo biloba extracts orisolated Ginkgolide B to said patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of apoptosis-related proteins Adenosine A2AReceptor, Flt3 ligand, Grb2, Clusterin, RXR-β, Glutathione S-transferaseP, N-Myc, TRADD, SGP-2 and NIP-1, in a patient in need of suchdecreasing, which comprises administering an effective amount of Ginkgobiloba extracts or isolated Ginkgolide B to said patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of transcription factors Id-2, ATF-4, ETR101and ETR-103 in a patient in need of such decreasing, which comprisesadministering an effective amount of Ginkgo biloba extracts or isolatedGinkgolide B to said patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of growth factors macrophagecolony-stimulating factor-1, heparin-binding EGF-like growth factor,hepatocyte growth factor-like protein and inhibin α, in a patient inneed of such decreasing, which comprises administering an effectiveamount of Ginkgo biloba extracts or isolated Ginkgolide B to saidpatient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of cell adhesion molecules CD19 B-lymphocyteantigen, L1CAM, β-catenin, integrin subunits α3, α4, α6, β5, and αM, ina patient in need of such decreasing, which comprises administering aneffective amount of Ginkgo biloba extracts or isolated Ginkgolide B tosaid patient.

In another aspect, the present invention is directed to a method ofdecreasing the expression of genes APC, PE-1, RhoA, c-Jun,prothymosin-α, CDK2, p55CDC, myeloblastin, p120 proliferating-cellnuclear antigen (PCNA), NET1, ERK2, Adenosine A2A Receptor, Flt3 ligand,Grb2, Clusterin, RXR-β, Glutathione S-transferase P, N-Myc, TRADD,SGP-2, NIP-1, Id-2, ATF-4, ETR-101, ETR-103, macrophagecolony-stimulating factor-1, heparin-binding EGF-like growth factor,hepatocyte growth factor-like protein, inhibin α, CD19 B-lymphocyteantigen, L1CAM, β-catenin, and integrin subunits α3, α4, α6, β5, and αM,in a patient in need of such decreasing, which comprises administeringan effective amount of Ginkgo biloba extracts or isolated Ginkgolide Bto said patient.

In another aspect, the present invention is directed to a pharmaceuticalcomposition comprising an effective amount of Ginkgo biloba extracts orisolated Ginkgolide B for combating cancer and a pharmaceuticallyacceptable carrier or diluent.

Of all of the foregoing methods and compositions of the presentinvention, a preferred embodiment of each is where the Ginkgo bilobaextracts is EGB 761®.

Further, of all the foregoing methods and compositions of the presentinvention, it is preferred that Ginkgolide B is used.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color(FIG. 11). This photograph is retained by the International Bureau aspart of the record copy.

FIG. 1. Effect of various concentrations of EGB 761® on MDA-231 PBRligand binding capacity. MDA-231 cells were cultured as described underMaterials and Methods. Cells were treated with the indicatedconcentrations of the injectable form of EGB 761®. At the indicated timeperiods cells were collected and PBR ligand binding characteristics weredetermined by Scatchard analysis. Data points represent the mean±S.D. ofthree independent experiments carried out in triplicate.

FIG. 2. Effect of various concentrations of GKB on MDA-231 PBR ligandbinding capacity. MDA-231 cells were cultured as described underMaterials and Methods. Cells were treated with the indicatedconcentrations of GKB for 48 hours. At the indicated time periods cellswere then collected and PBR ligand binding characteristics weredetermined by Scatchard analysis. Data points represent the mean±S.D. oftwo independent experiments carried out in triplicate.

FIG. 3. Effect of EGB 761® and GKB on PBR mRNA levels in MDA-231 cells.Cells were treated for 48 hours without or with either 20 (EGb-20) or200 (EGb-200) μg/ml EGB 761® or 2 (GKB-2) or 20 (GKB-20) μg/ml GKB. Atthe end of the incubation total RNA was isolated and loaded onto a 1%formaldehyde gel at a concentration of 10 μg/lane. Northern blots wereincubated with ³²P-labeled hPBR probe and exposed to XOMAT Kodak film.Top, autoradiogram of the blot. PBR migrates at 0.9 Kb. Bottom, relativeintensity of the PBR mRNA/28S ribosomal RNA visualized by ethidiumbromide staining. The autoradiogram and PBR mRNA quantitation representone out of two independent experiments.

FIG. 4. Effect of EGB 761 ® on MDA-231 cell proliferation. MDA-231 cellsgrown in 96-well plates were washed with PBS and cultured in mediasupplemented with 10% FBS in the presence or absence of the indicatedconcentrations of EGB 761®. 4 h prior to the end of incubation,bromodeoxyuridine (BrdU) was added to each well. Incorporation of BrdUwas measured at 450 nm (reference=700 nm). Data points represent themean±S.D. of four independent experiments carried out in quadruplicate.One-way ANOVA indicates that MDA-231 cell proliferation wassignificantly altered by treatment with EGB 761® at 48, 72 and 96 htimepoints (P<0.0001).

FIG. 5 Right, Middle, Left. Recovery of MDA-231 cell proliferation uponremoval of EGB 761®. MDA-231 cells grown in 96-well plates were washedwith PBS and cultured in media supplemented with 10% FBS in the presenceor absence of 2 (Left), 20 (Middle) or 200 (Right) μg/ml EGB 761® for 48h. At the end of the treatment the cells were washed and incubated inEGB 761®-free media for 48 h. 4 h prior to the end of incubation,bromodeoxyuridine (BrdU) was added to each well. Incorporation of BrdUwas measured at 450 nm (reference=700 nm). Data points represent themean±S.D. of two independent experiments carried out in quadruplicate.

FIG. 6. Effect of GKB on MDA-231 cell proliferation. MDA-231 cells grownin 96-well plates were washed with PBS and cultured in mediasupplemented with 10% FBS in the presence or absence of either 2 μg/mlor 20 μg/ml GKB for 48 hours. 4 h prior to the end of incubation,bromodeoxyuridine (BrdU) was added to each well. Incorporation of BrdUwas measured at 450 nm (reference=700 nm). Data points represent themean±S.D. of two independent experiments carried out in quadruplicate.One-way ANOVA indicates that MDA-231 cell proliferation wassignificantly altered by treatment with GKB (P<0.0001).

FIG. 7. Effect of EGB 761® and GKB on MCF-7 cell proliferation. MCF-7cells were grown in 96-well plates as described in FIG. 4 for theMDA-231 cells. MCF-7 cells were treated for 48 hours without or witheither 20 (EGb-20) or 200 (EGb-200) μg/ml EGB 761®, or 2 (GKB-2) or 20(GKB-20) μg/ml GKB. 4 h prior to the end of incubation,bromodeoxyuridine (BrdU) was added to each well. Incorporation of BrdUwas measured at 450 nm (reference=700 nm). Data points represent themean±S.D. of two independent experiments carried out in quadruplicate.One-way ANOVA indicates that MCF-7 cell proliferation was altered to alesser degree than the MDA-231 cell proliferation by treatment with EGB761® or GKB.

FIG. 8. Effect of EGB 761® on MDA-231 cell free radical production.MDA-231 cells were treated for 48 hours with 2 (EGb-2), 20 (EGb-20) or200 (EGb-200) μg/ml of EGB 761®. The cells were then washed and thelevels of cellular oxidative stress were measured using the fluorescentprobe DCF as described under Materials and Methods. Results shown aremeans±S.D. (n=4). Statistical analysis indicated that the effect of EGB761® was not significant.

FIG. 9. Transcriptional response to EGB 761® suggests an effect on genesinvolved in cell proliferation. Results shown represent quantitativeanalysis of the Atlas human cDNA expression array containing 588PCR-amplified cDNA fragments (Clontech Inc.). mRNAs were obtained fromcontrol or EGB 761® (20 μg/ml) treated, for 48 h, MDA-231 cells. Fornormalizing the mRNA abundance, the densitometric values obtained fromimage analysis were normalized using the housekeeping genes provided inthe array. Only consistent significant changes above 30% wereconsidered.

FIG. 10. Growth of MDA-231 xenografts in nude mice following either EGB761® or GKB treatments. Animals were treated either orally with 50 mg/kgEGB 761® or ip with 1 mg/kg GKB once a day for a month starting with100–150 mm³ volume MDA-231 tumors. After the end of the treatment theanimals were kept for 30 more days and then the animals were sacrificedon day 60. Data shown are means±S.E.M. (n=10). Statistical analysisindicated that the effects of EGB 761® and GKB were significant comparedto their respective controls (p<0.05).

FIG. 11 A, B, C, D, E, F, G, H. PBR expression in MDA-231 xenograftsfrom control and EGB 761® or GKB treated animals. Formalin embeddedsections of MDA-231 xenografts were immunostained with an anti-PBRantiserum at 1:500 dilution as described in the Materials and Methodssection. MDA-231 tumors were obtained from animals treated with vehicle(A–D), 50 mg/kg EGB 761® per os (E, G), or 1 mg/kg GKB ip (F, H). Ashows cells found in the middle of a tumor obtained from vehicle-treatedanimals where the nuclear localization of the PBR protein can be easilyseen (see arrowheads). B shows the immunostaining seen in cells presentin the edge of the tumor obtained from vehicle-treated animals. A highermagnification of the immunostaining seen in the cells present in theedge of the tumor obtained from vehicle-treated animals is shown in C. Drepresents a control treated with a non-specific antiserum. E shows thePBR immunostaining in cells found in the middle of the tumor obtainedfrom animals treated with EGB 761®. F shows the PBR immunostaining incells found in the middle of the tumor obtained from animals treatedwith GKB. G shows the PBR immunostaining in cells found in the edge ofthe tumor obtained from animals treated with EGB 761®. H shows the PBRimmunostaining in cells found in the middle of the tumor obtained fromanimals treated with GKB. Arrowheads indicate nuclei. Magnification is×75 for A, B, C, E, F, G, and H and ×150 for C.

DETAILED DESCRIPTION

The term “ginkgo terpenoid” includes all of the naturally occurringterpenes which are derived from the gymnosperms tree Ginkgo biloba aswell as synthetically produced ginkgo terpenoids and pharmaceuticallyactive derivatives and salts thereof and mixtures thereof. Examples ofginkgo terpenoids include ginkgolides. Examples of ginkgo terpenoids aredisclosed in Ginkgolides, Chemistry, Biology, Pharmacology, and ClinicalPerspectives, J. R. Provs. Science Publishers, Edited by P. Braguet(1988); F. V. DeFeudis, Ginkgo Biloba Extract (EGB 761®);Pharmacological Activities and Clinical Applications, Elsevier, ChapterII (1991).

The term “ginkgolide” as used herein include the various ginkgolidesdisclosed in the books cited above as well as non-toxic pharmaceuticallyactive derivatives thereof. Examples of ginkgolide derivatives includetetrahydro derivatives, acetyl derivatives, and alkyl esters such as themonoacetate derivatives and triacetate derivatives disclosed in Okabe,et al., J. Chem. Soc. (c), pp. 2201–2206 (1967). Ginkgolide B has thefollowing structure and as used herein, refers to isolated ginkgolide B:

The term “Ginkgo biloba extract” as used herein includes a collection ofnatural molecules, including terpenoids, derived from the leaves of theGinkgo biloba tree. Preferably, the extract is the specific formulationof Ginkgo biloba extract known as EGB 761®.

The term “combating” as used herein means preventing, inhibiting and ordecreasing whatever the word “combating” acts upon, e.g., combatingcancer cell proliferation means that the cancers cells are prevented andinhibited from proliferating further and or the degree or rate ofproliferation is decreased.

The level of expression of PBR, for the purposes of diagnosis orprognosis of a cancer or tumor, can be detected at several levels. Usingstandard methodology well known in the art, assays for the detection andquantitation of PBR RNA can be designed, and include northernhybridization assays, in situ hybridization assays, and PCR assays,among others. See e.g., Maniatis, Fitsch and Sambrook, MolecularCloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D.N. Glover ed. 1985), or Current Protocols in Molecular Biology, Ausubel,F. M. et al. (Eds), Wiley & Sons, Inc. for the general description ofmethods for nucleic acid hybridization. Polynucleotide probes for thedetection of PBR RNA can be designed from the sequence available ataccession number L21950 for the human PBR sequence (Riond, J. et al.(1991) Eur. J. Biochem. 195:305–311; Chang, Y. J. et al. (1992) DNA andCell Biol. 11:471–480). The sequence of PBR from other sources such asbovine (Parola, A. L. et al. (1991) J. Biol. Chem 266:14082–14087) andmouse (Garnier, M. et al. (1994) Mol Phar. 45:201–211) are also known.

The complete sequence of the PBR, normal or mutant, can be used for aprobe to detect RNA expression. Alternatively, a portion or portions ofthe sequence can be used. Methods for designing probes are known in theart. Polynucleotide sequences are preferably homologous to orcomplementary to a region of the PBR gene, preferably, the sequence ofthe region from which the polynucleotide is derive is homologous to orcomplementary to a sequence which is unique to the PBR gene. Whether ornot a sequence is unique to the PBR gene can be determined by techniquesknown to those of skill in the art. For example, the sequence can becompared to sequences in databanks, e.g., GenBank. Regions from whichtypical DNA sequences may be derived include but are not limited to, forexample, regions encoding specific epitopes, as well as non-transcribedand/or non-translated regions.

PBR ligands or anti-PBR antibodies, or fragments of ligand andantibodies capable of detecting PBR may be labeled using any of avariety of labels and methods of labeling for use in diagnosis andprognosis of disease, such as breast cancer, particularly for assayssuch as Positron Emission Tomography and magnetic resonance imaging(Leong, D. et al. (1996) Alcohol Clin. Exp. Res. 20: 601–605). Examplesof types of labels which can be used invention include but are notlimited to enzyme labels, radioisotopic labels, non-radioactive isotopiclabels and chemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateinsomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ³²P,³⁵S, 14C, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹Ci, ²¹¹At, ²¹²Pb,⁴⁷Sc, ¹⁰⁹Pd, ¹¹C, ¹⁹F, 123I, etc.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, ⁴⁶Fe, etc.

Examples of suitable fluorescent labels include a ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycodyanin label, an allophycocyanin label, afluorescamine label, etc.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, etc.

We examined herein the effect of Ginkgo biloba extracts, morespecifically EGB 761® and GKB on PBR expression and cell proliferation,particularly in human breast cancer cells. We used the highly aggressivecell line MDA-231, which expresses over 60-fold higher levels of PBRligand binding and mRNA relative to the non-aggressive cell line MCF-7.EGB 761® and GKB decreased in a time- and dose-dependent manner PBRexpression and cell proliferation in MDA-231 cells whereas EGB 761® andGKB did not affect the MCF-7 cell proliferation to the same degree. Thiseffect was reversible and it was not due to the antioxidant propertiesof the compounds tested.

The determination of elevated levels of PBR is done relative to a samplewith no detectable tumor. This may be from the same patient or adifferent patient. For example, a first sample may be collectedimmediately following surgical removal of a solid tumor. Subsequentsamples may be taken to monitor recurrence of tumor growth and/or tumorcell proliferation. Additionally, other standards may include cells ofvarying aggressive phenotype such that an increase or decrease inaggressive phenotype can be accessed.

The distinct sub-cellular localization of PBR in the cytoplasm ofepithelial cells of normal breast ducts and the absence of staining inthe nucleus and the perinuclear area of the aggressive tumor cellsprovides a simple method for diagnosing the aggressive phenotype of atumor cell. Immunostaining using labeled PBR ligand or labeled PBRantibody or fragment of ligand or antibody capable of binding to PBR anddetermining the sub-cellular location of PBR in the cellular samplesprovides yet another diagnostic assay of the present invention. Inaddition, antiserum which recognizes PBR can also be used along with asecondary antibody reactive with the primary antibody. Immunostainingassays are well known in the art, and are additionally described in theExamples below with respect to breast cancer cells and biopsies.

An increase in the level of PBR is determined when the level of PBR in atumor cell is about 2–3 times the level of PBR in the normal cell, up toabout 10–100 times the amount of PBR in a normal cell.

Cell Culture and Treatments. Human breast cancer cell lines (MCF-7 andMDA-231) were obtained from the Lombardi Cancer Center, GeorgetownUniversity Medical Center. Cell lines were cultured on polystyreneculture dishes (Corning) and grown in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum (FBS). The injectableform (IPS200) of the standardized Ginkgo biloba extract EGB 761® wasused. This injectable form is devoid of protocyanidins, which are knownto interact with proteins in vitro (Defeuder, 1998 Ullstein Medical).The injectable form of EGB 761® and GKB (BN 52021) isolated from EGB761® were provided by the Institut Henri Beaufour-IPSEN (Paris, France).Radioligand Binding Assays. Cells were scraped from 150 mm culturedishes into 5 ml phosphate buffered saline (PBS), dispersed bytrituration, and centrifuged at 500×g for 15 min. Cell pellets werere-suspended in PBS and assayed for protein concentration. [³H]PK 11195binding studies on 50 μg of protein from cell suspensions were performedas previously described (Papadopoulos, V. et al., 1990, J. Biol. Chem.265: 3772–3779; Hardwick, M. et al., 1999, Cancer Research, 59: 631–632)the contents of which are incorporated herein by reference.[N-methyl-3H]PK 11195(1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide;sp. Act. 83.50 (Ci/mmol), was obtained from Du Pont-New England Nuclear(Wilmington, Del.) and PK 11195 was obtained from Research BiochemicalsIncorporated (Natick, Mass.). Scatchard plots were analyzed by theLIGAND program (Munson, P J, and Robbard, D. 1980, Anal. Biochem., 107:220–239) (BIOSOFT, Ferguson, Mo.).Protein Measurement. Protein levels were measured by the Bradford method(Bradford, M M, 1976, Anal. Biochem., 72: 248–254) using the Bio-RadProtein Assay kit (Bio-Rad Laboratories, Hercules, Calif.) with bovineserum albumin as a standard.RNA (Northern) Analysis. PBR mRNA expression in MDA-231 cells treatedwith the various compounds was examined by Northern Blot analysis as wepreviously described (Hardwick, M., et al., 1999, Cancer Research, 59:831–842). In brief, total cellular RNA was isolated using the RNAzol Breagent (TEL-TEST, Inc., Friendswood, Tex.) and chloroform. 20 μg oftotal RNA from each cell line were run on 1% agarose gels andtransferred overnight to nylon membranes (S&S Nytran, Schleicher &Schuell, Keene, N. H.) (21). A 0.2 kb human PBR (hPBR) cDNA fragment(derived from the pCMV5-PBR plasmid vector containing the full lengthhPBR kindly given by Dr. Jerome Strauss, University of Pennsylvania, PA)was radiolabeled with [α-³²P]dCTP using a random primers DNA labelingsystem (Life Technologies, Gaithersburg, Md.). The hybridizationconditions were as we previously described (Hardwick, M., et al., 1999,Cancer Research, 59: 831–842). Autoradiography was performed by exposingthe blots to X-OMAT AR film (Kodak, Rochester, N.Y.) at −70° C. for 4–48hr. Quantification of PBR mRNA was carried out using the SigmaGelsoftware (Jandel Scientific, San Rafael, Calif.).Nucleic Acid Arrays. We used the Atlas human cDNA expression array Ifrom Clontech (Palo Alto, Calif.). This array contains 588 humanPCR-amplified cDNA fragments of 200–500 bp long immobilized on apositively charged nylon membrane. MDA-231 cells were treated with andwithout 20 μl/ml EGB 761® for 48 hours. Poly A+ RNA was isolated fromcontrol and EGB 761®-treated cells. ³²P-labeled cDNA probes weregenerated from each poly A+ RNA and hybridized to the Atlas arrayaccording to the manufacturer's recommendations. Autoradiography wasperformed by exposing the blots to X-OMAT AR film (Kodak, Rochester,N.Y.) at −70° C. for 4–96 hr. Quantification of the hybridization seenwas carried out using the SigmaGel software (Jandel Scientific, SanRafael, Calif.). Multiple exposures were used in order to detect genesexpressed at low levels. The three internal controls, ubiquitin, G3PDHand β-actin were used to compare the relative expression levels of thedetected gene products in the control and EGB 761®-treated cells.Experimental variations were corrected using the ratios of geneexpression versus the internal controls. The effect of the EGB 761®treatment on each gene product is expressed as % of control (untreated)cells. The results presented herein show genes affected consistently, ata level above 30% of control, by the EGB 761® treatment.BrdU Cell Proliferation Assays. MDA-231 cells were plated on 96-wellplates (Corning, Corning, N.J.) at a concentration of approximately10,000 cells/well (24 h incubation) or approximately 5,000 cells/well(48 h incubation) in DMEM supplemented with 0.1% FBS. The cells werethen incubated in 10% FBS with various concentrations of EGB 761® or GKBfor the indicated time periods. Differences in cell proliferation wereanalyzed by measuring the amount of 5-bromo-2′deoxyuridine (BrdU)incorporation determined by the BrdU ELISA (Boehringer Mannheim,Indianapolis, Ind.). Incorporation of BrdU was measured at 450 nm(reference at 690 nm).Analysis of oxidative stress. Levels of cellular oxidative stress weremeasured using the fluorescent probe 2,7-dichlorofluorescin diacetate(2,7-DCF; Molecular Probes, Inc., Eugene, Oreg.) as described inGoodman, Y. and Mattson, M. P., Exp. Neurol., 128: 1–12, 1994. In brief,cells were cultured in 96-well plates and treated for 48 hours with theindicated concentrations of EGB 761®. At the end of the treatment thecells were washed and incubated in the presence of 50 μM 2,7-DCF in PBS.Fluorescence was then quantified using the Victor² quantitativedetection fluorometer (EGG-Wallac, Inc., Gaithersburg, Md.).Biological Evaluation In Vivo. The MDA-231 human breast cancer (estrogeninsensitive) xenograft model was used for in vivo screening of EGB 761®and GKB. Based on the in vitro data and previously published in vivodata (23) the doses used were 50 mg/kg for the EGB 761® and 1 mg/kg forthe GKB. Female athymic nude mice (NCI/Charles River, Frederick, Md.)are injected subcutaneously with 8×10⁶ MDA-231 tumor cells and tumorsare allowed to form to a volume of ˜100 to 150 mm³. At this time, groupsof 10 animals per compound were injected either orally for the EGB 761®or intraperitoneally for the GKB once a day for a month. Twice weeklytumor sizes and body weights were recorded for all animals for the 30days of treatment as well for 30 days after the end of the treatment. Atthat time the animals were sacrificed and the tumors were removed andprocessed for immunohistochemistry. Animal care was in accordance withinstitutional guidelines.Immunocytochemistry of MDA-231 tumors. MDA-231 tumors removed from themice treated with or without EGB 761® or GKB were fixed in 10% bufferedformalin. Tumors were sectioned and then placed on glass slides andprocessed as we previously described (19). For immunohistochemistry withanti-PBR primary antibodies, tissue sections were treated with a 30%H₂O₂/methanol mixture (1:9 ratio) for 5 min at room temperature toneutralize endogenous peroxidase activity and then washed well with PBS.Primary antibody in 10% calf serum in PBS was added to sections at aconcentration of 1:500 at RT for 1 h. Secondary antibody reactions wereperformed using horseradish peroxidase-coupled goat anti-rabbitsecondary antibody diluted 1:500 in PBS supplemented with 10% calfserum. After washing the slides three times in PBS for 2 min each, freshH₂O₂ diluted 1:1,000 with 3-amino-9-ethyl carbazole (AEC) was added andslides were incubated for 1 h at 37° C. The slides were then rinsed indistilled H₂O before mounting with Crystal/Mount.Statistical Analysis. Comparison of multiple means was performed withInStat's one-way analysis of variance (ANOVA) (GraphPad Inc., San Diego,Calif.). All F statistics and P values for one-way ANOVAs are providedin the text. Comparison of individual drug treatments to the controltreatments was performed with unpaired t-test. All p values for unpairedt-tests are provided in the text.ResultsEGB 761® and GKB reduce the PBR Ligand Binding Capacity of the MDA-231Human Breast Cancer Cells. FIG. 1 shows that increasing concentrationsof the injectable form of EGB 761® inhibit in time-dependent manner thePBR ligand binding capacity (Bmax), determined using saturationisotherms with the radiolabeled ligand PK 11195 followed by Scatchardanalysis of the data. Similar results were obtained using isolated GKB(FIG. 2). Interestingly, EGB 761® and GKB decreased PBR levels by 66% ofcontrol values. No significant effects on the receptor affinity (Kd)could be seen (5.8±1.4 pmol/mg protein, n=12).EGB 761® and GKB reduce the PBR mRNA Expression in MDA-231 Human BreastCancer Cells. RNA (Northern) blot analysis was performed in order todetermine if the differences seen in PBR ligand binding between thecontrol and the EGB 761®- or GKB-treated cells reflect an effect on PBRmRNA expression. As shown in FIG. 3, both EGB 761® and GKB reduced PBRmRNA levels. This result fits with the results presented above on thePBR ligand binding expression.EGB 761® and GKB Inhibit MDA-231 Cell Proliferation. Using theBromodeoxyuridine (BrdU) Cell Proliferation ELISA (Boehringer-Mannheim,Indianapolis, Ind.), we examined the effect of increasing concentrationsof EGB 761® on MDA-231 cell proliferation. FIG. 4 shows that EGb-761inhibits in a concentration- and time-dependent manner the MDA-231 cellproliferation. This effect of EGB 761® was reversible, even for thehighest concentration of EGB 761® used. (FIG. 5). Incubation of MDA-231cells for 48 hours with EGB 761®, followed by washing and incubation foranother 48 hours in EGB 761®-free medium, resulted in the recovery ofthe MDA-231 proliferative activity. Increasing concentrations of GKBalso inhibited the MDA-231 cell proliferation after 48 hours treatment(FIG. 6).EGB 761® and GKB Do Not Have The Same Level of Inhibition Against MCF-7Cell Proliferation as They Do Against MDA-231 Cell Proliferation.Compared to the MDA-231 cells, the non-aggressive MCF-7 cells containextremely low (<60 fold) PBR levels, as determined by both ligandbinding studies and mRNA analyses, we examined whether EGB 761® and GKBaffect the MCF-7 cell proliferation rate to the same degree as itaffects MDA-231 cell proliferation rate. FIG. 7 clearly shows thatneither EGB 761® nor GKB affect MCF-7 cell proliferation to the samedegree as they affect the MDA-231 cell proliferation.EGB 761® Does Not Affect MDA-231 Free Radical Levels. Recent in vivo andin vitro studies demonstrated that the terpene constituents of EGB 761®,including GKB, have anti-oxidant properties. In order to determinewhether the anti-proliferative effect of EGB 761® was due to itsanti-oxidant properties, we determined the free radical levels inMDA-231 cells treated with and without increasing concentrations of EGB761® (FIG. 8). Although a 20% decrease in free radical levels could beseen, this effect was neither statistically significant nordose-dependent, indicating that the effect seen was not due to an EGB761®-induced decrease of free radical levels in the cells.EGB 761® Regulates the MDA-231 Transcriptional Program Related to CellProliferation. The results presented above indicate that EGB 761® andGKB inhibit PBR expression and cell proliferation in the PBR-rich andhighly aggressive MDA-231 breast cancer cells. The non-aggressive MCF-7cells, which contain extremely low PBR levels, did not respond to EGB761® treatment to the same degree as that of the MDA-231 breast cancercells. In order to determine whether the effect of EGB 761® (20 μg/mlfor 48 hours) on MDA-231 cells was specific for PBR or whether othergenes involved in cell proliferation were affected by the treatment, weused a cDNA array representing 588 distinct human genes. As noted underMaterials and Methods, experimental variations were corrected using theratios of gene expression versus the internal controls. The effect ofthe EGB 761® treatment on each gene product is expressed as % of control(untreated) cells. Only consistent changes above 30% of control valuesare presented in FIG. 9 and Table I.Biological Evaluation of the Effect of EGB 761® and GKB in Vivo In orderto assess the effect of EGB 761® and GKB on tumor cell proliferation andPBR expression in an in vivo setting we used the mammary fat padxenograft implantation model (See Medina, D., J. Mamm. Gland. Biol.Neopl. 1:5–19, 1996). FIG. 10 shows that 30 days treatment with either50 mg/kg EGB 761® or with 1 mg/kg GKB resulted in a 35% (p=0.037) and32% (p=0.043) decrease in tumor size, measured a month after the end ofthe treatment, respectively. These treatments did not affect the animalbody weight (data not shown). Considering these in vitro data on theeffect of EGB 761® and GKB on PBR expression in MDA-231 cells, weexamined whether EGB 761® and GKB also decreased PBR expression in theMDA-231 xenografts. FIG. 11(A–D) shows horseradish peroxidase (HRP)staining of the PBR antiserum used to detect the 18,000 molecular weightprotein in MDA-231 xenografts from vehicle-treated animals. Thehematoxylin counterstain was omitted in order to distinguish the nuclearlocalization of PBR (19) in the tumors. FIG. 11A shows the middle of atumor where the nuclear localization of the 18,000 PBR protein can beeasily seen (see arrowheads). FIG. 11B shows the immunostaining seen inthe edge of the tumor obtained from vehicle-treated animals. A highermagnification of the immunostaining seen in the edge of the tumorobtained from vehicle-treated animals is shown in FIG. 1C, and a controltreated with a non-specific antiserum is shown in FIG. 11D. Treatmentwith either EGB 761® (FIG. 11E) or GKB (FIG. 11F) reduced the nuclearPBR expression present in cells found in the middle of the tumor.Interestingly, treatment with either EGB 761® (FIG. 11G) or GKB (FIG.11H) also eliminated the nuclear PBR expression present in the cells atthe edge of the tumors. However, in the later case cytosolicimmunostaining could be seen (FIGS. 11G and H). These data werereplicated in sections taken from xenografts grown in three separateanimals.

It is of interest to note that even in the presence of highconcentrations of either EGB 761® or GKB, PBR levels and rates of cellproliferation could not be reduced below 30% of normal values. Thissuggests that there is a minimum of PBR required to maintain membraneintegrity and cell function. It should be also noted that even at thehighest concentrations used, neither EGB 761® nor GKB were toxic for thecells, because cell proliferation recovered upon removal of thecompounds. These data suggest that these compounds are cytostatic andnot cytotoxic. Additional cytotoxicity assays indicated that under thesame conditions neither EGB 761® nor GKB induced any significant celldeath.

The absence of any significant decrease in the amount of reactive oxygenspecies produced in the MDA-231 cells by EGB 761® or GKB suggests thattheir anti-oxidant properties were not responsible for decreasing PBRexpression and cell proliferation in the MDA-231 cells. These resultsindicate that these compounds may regulate PBR gene transcription eitherdirectly or indirectly.

The finding that EGB 761® and GKB decreased PBR expression and cellproliferation in the highly aggressive, nuclear PBR-expressing MDA-231cells, but did not affect the non-aggressive MCF-7 cells, which haveextremely low PBR levels and are devoid of nuclear PBR, to the samedegree as they did for MDA-231 cells, provides an additional support tothe hypothesis that the presence of PBR may be a determinant factor forthe aggressive phenotype of breast tumor cells. Moreover, thisobservation further demonstrates the specificity of the effect of EGB761® and GKB on targeting the regulation of PBR expression. This laterfinding brought us to two key questions raised by the current study: isthe expression of other genes regulated by EGB 761®? is there atranscriptional program activated or inhibited by EGB 761®, where PBR isat the origin or a part of a cascade of events, responsible for alteringthe proliferation rate of MDA-231 cells? To address these questions, weutilized the Atlas Human cDNA Expression Array. As indicated in Table I,treatment of MDA-231 cells with the EGB 761® extract induced alterationsin the transcriptional expression of 36 out of 588 genes examined. Notsurprisingly, the vast majority of the affected genes have close ties toeither cell proliferation, differentiation, or apoptosis. Perhaps themost telling of the effects EGB 761® has on the MDA-231 cell line is thedown-regulation of the p120 proliferation-cell nuclear antigen. p120 isused as a prognostic indicator in breast cancer patients and prostateadenocarcinomas (Perlaky, L., et al., Cancer Res., 52: 428–436, 1992;Zhuang, S. H. et al., Endocrinology, 139: 1197–1207, 1998). Moreimportantly, however, p120 is an immunocytochemical marker ofproliferating cells. Down-regulation of this proliferation marker by 68%thus confirms our data demonstrating that EGB 761® inhibits MDA-231 cellproliferation.

Using a human cDNA expression array we examined the effect of the EGB761 treatment on the expression of 588 genes in MDA-231 cells. We foundthat the treatment increased the expression of the c-Myc protooncogeneand decreased the expression of 35 gene products, including oncogenes(AP-1, PE-1, RhoA, n-Myc), cell cycle regulators (CDK2, p55CDC, PCNAp120), signal transduction modulators (NET1, ERK2), apoptosis-relatedproducts (SGP-2, NIP1) receptors (A2A, RXR-beta, Grb2), transcriptionfactors (Id-2, ATF4, ETR101, ETR-103), growth factors (HB-EGF,HGF-like), and cell adhesion molecules (CD19, L1CAM, integrins α3, α4,α6, β5, Mac-1, β-catenin) which are directly involved in variouspathways regulating cell proliferation. Considering that the compoundstested were effective only on the MDA-231-cells, which express highlevels of PBR, these data suggest that the expression of nuclear PBR maybe a determining factor for a tumor cell to acquire an aggressive andinvasive phenotype.

TABLE I Effect of EGB 761 ® on MDA-231 gene expression examined usingthe Atlas human cDNA expression array as described under Materials andMethods. % Name Change Function References Oncogenes and TumorSuppressers c-Myc +75% basic helix-loop-helix-leucine zippertranscription factor (37) Myc/Max heterodimers induce cell-cycleprogression, apoptosis, and malignant transformation c-Jun −78% part ofthe AP-1 transcription factor that regulates genes involved (38) in cellproliferation RhoA −93% GTP-binding protein that is an importantregulator of cell (39) proliferation RhoA inactivation inhibits HL60cell proliferation (40) APC −59% APC mutations are associated with bothhereditary and sporadic (41) colorectal cancers a negativepost-translational regulator of β-catenin (42) PE-1 −42% transcriptionfactor (43) Cell Cycle Control Proteins Prothymosin-α −79% acidicnuclear protein that is upregulated in proliferating (44) thymocytes,lymphocytes from leukemia patients, and in malignant breast lesionsMyeloblastin −66% a serine protease involved in leukemia celldifferentiation (45) p55CDC −63% similar to mitosis regulators CDC4 andCDC20 (46) expression positively correlated with cell proliferationstatus p120 Proliferating-cell −68% nucleolar protein expressed inproliferating cells (47) Nuclear Antigen a prognostic indicator forbreast cancer patients and prostate (48) adenocarcinoma CDK2 −83%cyclin-dependent tyrosine kinase involved in progression through (49)the cell cycle cyclin E/Cdk2 inactivates the retinoblastoma tumorsuppresser to (50) allow the cell to progress to S phase Vitamin Dinhibition of LNCaP cell proliferation coincided with a reduction inCdk2 activity Intracellular Transducers NET1 −55% RhoA-specific guanineexchange factor (51) NIH3T3-transforming protein ERK2 −46% member of theextracellular signal-related protein kinase family (52) activated uponcell stimulation Apoptosis-Related Proteins Adenosine A2A −40% Gprotein-coupled receptor involved in the cAMP signaling (53) Receptorpathway Flt3 ligand −58% ligand for the Flt3 cytokine receptor tyrosinekinase (54) induces proliferation of leukemic myeloid cells Grb2 −70% anadapter protein that links receptor tyrosine kinases to the (55)Ras/MAPK signaling pathway via its SH2 domain Clusterin −54% aglycoprotein associated with cell adhesion and apoptosis (56, 57)increased expression is linked to Alzheimer's disease (58) RXR-β −55%retiniod-activated transcription factor (59) inhibition of chondrocyteproliferation by retinoic acid causes a (60) reduction in RXR-β mRNAexpression Glutathione S- −39% a multi-drug resistance gene that isoverexpressed in various (61, 62) transferase P human tumors chemicalinhibition of GST-P inhibits proliferation of Jurkat T cells (63) N-Myc−74% c-myc family member (64) associated with early-onset retinoblastomaTRADD −51% TNFR-associated death domain protein (65) involved inTNFR-induced cell growth and differentiation NIP-1 −40% originallydescribed as a yeast nuclear transport protein (66) part of thetranslation initiation factor 3 (elF3) core complex (67)DNA-Binding/Transcription Factors Id-2 −65% a member of the Idhelix-loop-helix family of transcriptional (68) inhibitors involved inproliferation of human pancreatic cancer cells ATF4 −42% a member of theATF/CREB family of transcription factors (69) regulates Ras-inducedtransformation of NIH3T3 cells ETR103 −65% a macrophage-associatedimmediate early gene (70) ETR101 −60% a lymphocyte-associated immediateearly gene (71) Cell Surface Antigens and Adhesion Molecules CD19B-lymphocyte −62% B-lymphocyte integral membrane protein (72) Antigenexpression is down-regulated during retinoid-inhibition oflymphoblastoid B-cell proliferation L1CAM −72% neural cell adhesionmolecule (73) increased L1CAM expression is associated with high-grademigration of glioma cells β-catenin −58% involved in cadherin-mediatedcell-cell interactions (74) interacts with the TCF/LEF transcriptionfactors in the Wnt signaling pathway Integrin Subunits αM −41% mediatescellular adherence of human neutrophils with LFA-1β (75) α subunit ofthe elastase receptor β5 −55% β subunit of the vitronectin receptor (VR)(76) involved in cessation of oligodendrocyte proliferation (77)involved in murine retinal angiogenesis (78) α4 −49% cross-linking α4integrins inhibits LB lymphoma cell proliferation (79) also involved inmetastasis of melanoma and lymphoma cells (80) α3 −77% a functionallyperturbing α3 integrin antibody inhibits human (81) epithelial cellproliferation α6 −53% overexpression of α6 integrin collaborates withErbB2 to induce a (82) more malignant phenotype in NIH3T3 cellsExtracellular Signaling/Communication Proteins Macrophage Colony- −31%regulates the proliferation, differentiation, and survival of (83)stimulating Factor-1 monocytes, macrophages and their precursors (CSF-1)initiates a mitogenic signal that is required throughout G1 phase (84)CSF-1 stably transfected ovarian granulosa cells exhibit enhanced cellproliferation Heparin-binding EGF- −62% overexpressed in numerous humanglioma cell lines and in a (85) like Growth Factor (HB- majority ofglioblastomas EGF) stimulates human glioma cell proliferation HepatocyteGrowth −81% a transmembrane protein tyrosine kinase found to be (86)Factor-like Protein overexpressed in hepatoblastoma and in human primaryliver (HGFLP) carcinoma induces proliferation and migration of murinekeratinocytes (87) Inhibin α −69% a member of the inhibin family ofheterodimeric growth factors (88) inhibin α is a marker of trophoblasticneoplasia and is highly (89) expressed in virilizing adenomas

Two members of the Myc family of transcription factors, c-Myc and n-Myc,were found to be grossly altered in this experiment. Expression of theproto-oncogene c-Myc was increased by 75% while expression of n-Myc wasreduced by 74%. Both of these genes are overexpressed in several cancertypes (Kim, C. J., et al., Virchows Arch., 434: 301–305, 1999; Dang, C.V., Mol. Cel. Biol., 19: 1–11, 1999) and are strongly correlated withtumor cell proliferation. Previous studies have shown that arrest ofneuroblastoma cell growth by the tyrosine kinase inhibitor genistein isaccompanied by down-regulation of n-Myc expression. This data fitsextremely well with our cell proliferation and n-Myc data.Overexpression of c-Myc, however, is associated with a stimulation ofcell proliferation in normal serum conditions. Overexpression of c-Mycinduces cell death in the absence of serum or other survival factors.Taken together the data implies that deregulation of c-Myc expressionrequires the altered expression of other genes, as well.

In our microarray experiment, we discovered the deregulated expressionof several other c-Myc-related genes. One such gene, prothymosin a (proTα), is induced by c-Myc. However, expression of proT α is reduced by 79%rather than increased, as might be predicted by up-regulation of c-Myc.Further, expression of other c-Myc target genes such as cdc25A, cyclinA, and cyclin E are unaffected by treatment of MDA-231 cells with EGB761®, suggesting either a treatment-specific or a cell line-specificshort circuit in c-Myc-regulated gene transcription. Other data gatheredfrom the microarray experiment further supports this hypothesis. c-Myctranscriptional regulation is under the control of APC and β-catenin.However, both of these genes are down-regulated by EGB 761® in MDA-231cells (59 and 58%, respectively) while c-Myc is up-regulated. While someof this data appears to be contradictory, much of the published data onthe role of c-Myc in cell proliferation, differentiation, and cell deathalso appears contradictory.

Similar to the altered regulation of c-Myc and Myc-related proteins byEGB 761®, the microarray experiment exposed disruption of severalsignaling molecules. EGB 761® treatment resulted in a 93% reduction inthe expression of RhoA, a gene encoding a GTP-binding protein involvedin numerous cellular phenomena, and a 55% reduction in NET1 expression,a RhoA-specific guanine exchange factor. Interestingly, RhoA has beendemonstrated to regulate cyclin E/Cdk2 activity in fibroblasts.Activation of cyclin E/Cdk2 complex is crucial to the progression of thecell cycle from G1 to S-phase. Regulation of cyclin E/Cdk2 activity hasalso been demonstrated by c-Myc. Although the significance of these twophenomena is not immediately obvious, it should be noted that expressionof Cdk2 is reduced by 83% by EGB 761®.

Other important signaling molecules are also down-regulated by EGB 761®.Expression of the adapter molecule Grb2 is reduced by 70%. Grb2 plays animportant role in cellular signaling by physically linking signaltransducers such as receptor tyrosine kinases to the Ras/MAPK pathway.With regard to the MAPK pathway, expression of the MAP/ERK family memberERK2 is down-regulated by 46% and expression of the c-Jun transcriptionfactor is reduced by 78%. Interestingly, it has been reported that EGB761® is a suppressor of AP-1 transcription factor stimulated by phorbolesters. These data imply that the effects of EGB 761® on MDA-231 cellproliferation are accompanied by a broad reduction in mRNAs withfunctional relationships with one another.

Another interesting finding from the microarray experiment is thereduced expression of several integrins. The integrins are a largefamily of cell-cell and cell-extracellular matrix adhesion receptorsthat are composed of two transmembrane glycoprotein subunits, one α-andone β-subunit. All of the integrins represented in Table I are involvedin the regulation of cell proliferation in some way. For example,integrin αM is part of the elastase receptor. ONO-5046, an elastaseinhibitor, suppresses the proliferation of polyoma virus- and Kirstensarcoma virus-transformed BALB/c3T3 cells and of Capan-1 pancreaticcarcinoma cells. Moreover, integrin α4 has been implicated in theaccumulation of distal metastases in melanoma, sarcoma, and lymphomacell models. It is important to emphasize that integrins function asheterodimers of one α- and one β-subunit. Reduced expression of eitherthe α- or the β-subunit is clearly important in the regulation ofintegrin receptor function.

The expression of some key growth factor genes, such as the hepatocytegrowth factor-like and EGF-like growth factor, were also reduced by EGB761®. It is possible that the EGB 761®-induced inhibition of cellproliferation may be due to the reduced expression of these growthfactors that may act as autocrine regulators of cell growth.

EGB 761® and GKB are intended to be provided to recipient patient in anamount sufficient to combat cancer in said patient or in an amountsufficient to affect the expression (negatively or positively) of thegene products listed in Table 1, hereinabove. An amount is said to besufficient to combat cancer if the dosage, route of administration, etc.of the EGB 761® and GKB are sufficient to influence such a response.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

EGB 761® and GKB can be formulated according to known methods to preparepharmaceutically useful compositions, whereby these materials, or theirfunctional derivatives are optionally combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in Remington's PharmaceuticalSciences (16^(TH) ED., Osol, A. ed., Mack Easton Pa. (1980)). In orderto form a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe above-described compounds together with a suitable amount of carriervehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate) microcapsules, respectively, or in colloidal drugdelivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

EGB 761® and isolated GKB can be administered by oral, parenteral (e.g.,intramuscular, intraperitoneal, intravenous or subcutaneous injection,or implant), nasal, vaginal, rectal, sublingual or topical routes ofadministration and can be formulated with pharmaceutically acceptablecarriers to provide dosage forms appropriate for each route ofadministration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than suchinert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets and pills, the dosage forms may alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

The dosage of EGB 761® or isolated GKB in the compositions of thisinvention may be varied; however, it is necessary that the amount of theactive ingredient be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment. The dosecan be administered as a single dose or divided into multiple doses. Aneffective dose amount of either EGB 761® or isolated GKB depends uponthe condition being treated, the route of administration chosen andultimately will be decided by the attending physician or veterinarian.

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The contents of the publications and patents referenced herein areincorporated herein by reference in their entirety.

1. A method of inhibiting the proliferation of breast cancer cells in apatient in need of such inhibition, wherein said breast cancer cells arecharacterized by increased expression of peripheral-type benzodiazepinereceptor protein, which comprises: determining whether said breastcancer cells exhibit elevated expression of peripheral-typebenzodiazepine receptor protein, wherein said elevated expression is anat least 3-fold increase in the level of expression of peripheral-typebenzodiazepine receptor protein as compared to normal cells; andadministering an effective amount of isolated Ginkgolide B to saidpatient.
 2. A method according to claim 1, wherein said proliferation ofbreast cancer cells is caused by the over-expression of oncogenes, andwherein the administering results in decreasing the expression of saidoncogenes and combats the proliferation of said breast cancer cells. 3.A method according to claim 2, wherein said oncogenes are one or more ofAPC, PE-1, RhoA and c-Jun.
 4. A method according to claim 1, whereinsaid administering results in decreasing the expression ofperipheral-type benzodiazepine receptor in said breast cancer cells. 5.A method according to claim 4, wherein said breast cancer cells arehuman breast cancer cells.
 6. A method according to claim 4, wherein thedecreasing of the expression of peripheral-type benzodiazepine receptoris the result of decreasing the expression of peripheral-typebenzodiazepine receptor mRNA in said breast cancer cells.
 7. A methodaccording to claim 1, wherein said administering results in increasingthe expression of a c-Myc protooncogene.
 8. A method according to claim1, wherein said administering results in decreasing the expression ofcell cycle regulators prothymosin-α, CDK2, p55CDC, myeloblastin and p120proliferating-cell nuclear antigen.
 9. A method according to claim 1,wherein said administering results in decreasing the expression ofintracellular signal transduction modulators NET1 and ERK2.
 10. A methodaccording to claim 1, wherein said administering results in decreasingthe expression of apoptosis-related products Adenosine A2A Receptor,Flt3 ligand, Grb2, Clusterin, RXR-β, Glutathione S-transferase P, N-Myc,TRADD, SGP-2 and NIP-1.
 11. A method according to claim 1, wherein saidadministering results in decreasing the expression of transcriptionfactors Id-2, ATF-4, ETR101 and ETR-103.
 12. A method according to claim1, wherein said administering results in decreasing the expression ofgrowth factors macrophage colony-stimulating factor-1, heparin-bindingEGF-like growth factor, hepatocyte growth factor-like protein andinhibin α.
 13. A method according to claim 1, wherein said administeringresults in decreasing the expression of cell adhesion molecules CD19B-lymphocyte antigen, L1CAM, β-catenin, integrin subunits α3, α4, α6,β5, and αM.
 14. A method according to claim 1, wherein saidadministering results in decreasing the expression of genes APC, PE-1,RhoA, c-Jun, prothymosin-α, CDK2, p55CDC, myeloblastin, p120proliferating-cell nuclear antigen, NET1, ERK2, Adenosine A2A Receptor,Flt3 ligand, Grb2, Clusterin, RXR-β, Glutathione S-transferase P, N-Myc,TRADD, SGP-2, NIP-1, Id-2, ATF-4, ETR-101, ETR-103, macrophagecolony-stimulating factor-1, heparin-binding EGF-like growth factor,hepatocyte growth factor-like protein, inhibin α, CD19 B-lymphocyteantigen, L1CAM, β-catenin, and integrin subunits α3, α4, α6, β5, and αM.